Agrobacterium tumefaciens Mediated Transformation of Trichoderma reesei and Optimization of the Transforming Conditions

Objective:Transformation of the filamentous fungus Trichoderma reesei was achieved using Agrobacterium tumeficiens-mediated transformation(ATMT) method,and the factors influencing transformation efficiency were evaluated.Method:The binary vector pCAM-hph containing the hygromycin phosphotransferase(hph) gene was constructed based on a commercial vector pCAMBIA 0380,and was transformed into A.tumeficiens LBA4404.The A.tumeficiens transformants were co-cultivated with the T.reesei conidial spores,and the fungal transformants were selected on PDA plate containing 100μg/mL hygromycin.The DNA fragment that was inserted into the fungal transformants was analyzed through PCR amplification and DNA sequencing.Result:ATMT method was effectively applied in transformation of T.reesei,with a transformation efficiency of obtaining 25.8 transfotmats per 106 conidial spores.Acetosyringone concentration,the concentrations of the bacterial cells and the conidial spores,period of co-cultivation,and pH of the medium in co-cultivation evidently affected the efficiency of transformation.The optimal transformation conditions were: the concentration of the bacterial cells was OD660 0.8,the concentration of conidial spores was 106/ml,the co-cultivation period was 48 hours,the pH of co-cultivation was 5.0~5.5,and the culture temperature was 28℃.Conclusion:The ATMT method for transformation of T.reesei was constructed,and the transformation condition was optimized.